Redirection of the Respiro-Fermentative Flux Distribution in Saccharomyces cerevisiae by Overexpression of the Transcription Factor Hap4p

Abstract

Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae. Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.Modification and control of metabolic fluxes is an important goal in most industrial applications of microorganisms. This is the case for baker's yeast, which has applications in production of heterologous proteins, brewing, and baking. Maximization of biomass yields of baker's yeast growing on sugars is hampered by the cell's strong tendency to produce ethanol, even under aerobic conditions when sugars are present in excess. This alcoholic fermentation might be prevented by manipulation of the carbon flux distribution between fermentation and respiration.Attempts at redirection of carbon fluxes by interference in expression levels of single enzymes of glycolytic or fermentative pathways have so far not been successful (24,25). Reduced activities of fermentative enzymes such as pyruvate decarboxylase result in impaired growth on glucose (8, 9) and deprive the cells of their fermentative capacity necessary for raising of dough. Increasing respiratory activity seems to be a better approach, but numerous studies have shown that overproduction of a single enzyme results in either little or no increase of flux through a metabolic pathway (20). This is because flux control is usually not exerted by only a single enzyme (2,20,28). A more rational approach would therefore be to manipulate the activity of a regulatory protein involved in control of all key enzymes of one or more specific metabolic pathways. The potential of this approach is illustrated by the partial alleviation of glucose control of sucrose and galactose metabolism as a result of disruption of the glucose repressor Mig1p alone or in combination with Mig2p (16,17). These modifications do not, however, result in a significant change in respiratory functions, ethanol production, or biomass yield.Aiming at redirection of fermentative flux toward oxidative carbon flux, we...

Dissection of the promoter of the HAP4 gene in S. cerevisiae unveils a complex regulatory framework of transcriptional regulation

“…Transcriptional activation by the HAP complex during growth on non-fermentable carbon sources is hence dependent on the availability of Hap4p. A yeast strain overexpressing HAP4 showed partial alleviated repression of respiratory function and altered the fermentative metabolism on glucose towards a more oxidative metabolism (Blom et al, 2000). This confirms the view that during growth on glucose the level of Hap4p becomes limiting, leading to downregulation of the target genes, and shows the pivotal role Hap4p plays in the balance between oxidative and fermentative metabolism.…”

Reduced Histone Expression or a Defect in Chromatin Assembly Induces Respiration

“…The activation domain of the complex is contained within the Hap4p subunit (64). HAP4 expression increases upon glucose depletion (65), and overexpression of HAP4 induces respiration, even in a glucose-repressed state (53,66). Since the induction of Hap2/3/4/5p complex-responsive genes is independent of SNF1 activity, we decided to test the possibility that decreased expression of histone genes induces HAP4 expression.…”

Osmotic Stress Signaling and Osmoadaptation in Yeasts

“…Hap4p is part of the HAP complex, which activates expression of genes encoding tricarboxylic acid cycle and respiratory enzymes (138). Ectopic overexpression of HAP4 has been shown to stimulate oxidative catabolism of glucose and increases the biomass yield, indicative of more effective energy generation (44). Therefore, enhanced expression of HAP4 and genes encoding mitochondrial enzymes may result in the stimulation of oxidative metabolism in order to meet the energy demand during growth under these conditions.…”